1. Field of the Invention
The present invention relates to novel proteins activating pro-phenoloxidase (pro-PO) system of Tenebrio molitor, genes encoding the same, methods of detecting bacterial infection in a sample using the proteins, and kits for detecting bacterial infection in a sample using the proteins. The present invention also relates to a method of preparing a soluble linearized Lys-type peptidoglycan (SLPG), useful for a standard substance for the kit.
2. Description of Related Art
Recent genetic studies revealed that Drosophila melanogaster peptidoglycan (PG) recognition protein Drosophila PGRP-SA and Drosophila PGRP-SD activate the Toll pathway (Michel, T., Reichhart, J. M., Hoffmann, J. A. & Royet, J. (2001) Nature 414, 756-759; and Bischoff, V., Vignal, C., Boneca, I. G., Michel, T., Hoffmann, J. A. & Royet, J. (2004) Nat Immunol 5, 1175-1180), while Drosophila PGRP-LC and Drosophila PGRP-LE are receptors for the Imd pathway (Gottar, M., Gobert, V., Michel, T., Belvin, M., Duyk, G., Hoffmann, J. A., Ferrandon, D. & Royet, J. (2002) Nature 416, 640-644; Choe, K. M., Werner, T., Stoven, S., Hultmark, D. & Anderson, K. V. (2002) Science 296, 359-362; and Takehana, A., Katsuyama, T., Yano, T., Oshima, Y., Takada, H., Aigaki, T. & Kurata, S. (2002) Proc Natl Acad Sci USA 99, 13705-13710). The immune phenotype of a loss-of-function mutant of Drosophila Gram-negative bacteria binding protein 1 (Drosophila GNBP1) was indistinguishable from that of Drosophila PGRP-SA, demonstrating that these two proteins are required to activate the Toll pathway in response to Gram-positive bacterial infection (Gobert, V., Gottar, M., Matskevich, A. A., Rutschmann, S., Royet, J., Belvin, M., Hoffmann, J. A. & Ferrandon, D. (2003) Science 302, 2126-2130; Pili-Floury, S., Leulier, F., Takahashi, K., Saigo, K., Samain, E., Ueda, R. & Lemaitre, B. (2004) J Biol Chem 279, 12848-12853; and Wang, L., Weber, A. N., Atilano, M. L., Filipe, S. R., Gay, N. J. & Ligoxygakis, P. (2006) EMBO J 25, 5005-5014). However, the molecular mechanisms of the upstream part of the Toll pathway in Gram-negative bacteria recognition still remain to be elucidated.
The pro-phenoloxidase (pro-PO) activation cascade, which leads to melanization of invading microbes, is another major innate immune defense mechanism in invertebrates that is triggered by peptidoglycan (PG) and β-1,3-glucan (Cerenius, L. & Soderhall, K. (2004) Immunol Rev 198, 116-126; and Kanost, M. R., Jiang, H. & Yu, X. Q. (2004) Immunol Rev 198, 97-105). The pro-PO cascade, like the vertebrate complement system, is a proteolytic cascade in blood plasma. Therefore, the pro-PO system is an ideal tool for biochemical studies of PG and β-1,3-glucan recognition and subsequent signaling under a cell-free condition. We previously identified the Tenebrio molitor PGRP that exhibited the highest sequence homology with Drosophila PGRP-SA. This PGRP, which we designate Tenebrio PGRP-SA, activated the Lys-PG-dependent pro-PO system in Tenebrio beetle. Remarkably, a novel synthetic Lys-PG fragment functions as a competitive inhibitor of soluble polymeric linear Lys-PG in the activation of the pro-PO system. The synthetic Lys-PG fragment (hereinafter referred to as “synthetic muropeptide dimmer”), having a chemical structure of the following formula (I), is composed of tetra-saccharide (GlcNAc-MurNAc-GlcNAc-MurNAc), covalently linked to two copies of a tetrapeptide stem [L-Ala-D-isoGln-L-Lys-D-Ala) (Park, J. W., Je, B. R., Piao, S., Inamura, S., Fujimoto, Y., Fukase, K., Kusumoto, S., Soderhall, K., Ha, N. C. & Lee, B. L. (2006) J Biol Chem 281, 7747-7755).

Recent crystallographic structural studies of PGRP proteins without PG-fragment or in complex with PG-fragments provided important insights into the structural basis for PG recognition (Lim, J. H., Kim, M. S., Kim, H. E., Yano, T., Oshima, Y., Aggarwal, K., Goldman, W. E., Silverman, N., Kurata, S. & Oh, B. H. (2006) J Biol Chem 281, 8286-8295; Chang, C. I., Chelliah, Y., Borek, D., Mengin-Lecreulx, D. & Deisenhofer, J. (2006) Science 311, 1761-1764; Chang, C. I., Ihara, K., Chelliah, Y., Mengin-Lecreulx, D., Wakatsuki, S. & Deisenhofer, J. (2005) Proc Natl Acad Sci USA 102, 10279-10284; Guan, R., Roychowdhury, A., Ember, B., Kumar, S., Boons, G. J. & Mariuzza, R. A. (2004) Proc Natl Acad Sci USA 101, 17168-17173; Kim, M. S., Byun, M. & Oh, B. H. (2003) Nat Immunol 4, 787-793; and Chang, C. I., Pili-Floury, S., Herve, M., Parquet, C., Chelliah, Y., Lemaitre, B., Mengin-Lecreulx, D. & Deisenhofer, J. (2004) PLoS Biol 2, E277). Muropeptide, composed of N-acetylglucosamine and N-acetylmuramic acid sugars linked with a short peptide chain as a stem, was revealed as the minimum binding unit for PGRP-SA.
However, it remains unclear how recognition signal of Lys-PG by PGRPs primes the serine protease (SP) cascade leading to activation of the pro-PO or Toll pathways.